Published online before print January 18, 2008
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* Department of Pharmacology, Faculty of Medicine of Ribeirão Preto, University of Sao Paulo, Ribeirão Preto, SP, Brazil;
Laboratory of Molecular Biology, University of Uberaba, Uberaba, Brazil;
Division of Immunology and Endocrinology, National Institute for Biological Standards and Control, Potters Bar, United Kingdom; and
Departamento de Bioquímica e Imunologia, Instituto Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil
1 Correspondence: Faculdade de Medicina de Ribeirão Preto, USP, Avenida Bandeirantes, 3900, 14049-900, Ribeirão Preto, SP, Brazil. E-mail: fdqcunha{at}fmrp.usp.br
ABSTRACT
Neutrophil migration is responsible for tissue damage observed in inflammatory diseases. Neutrophils are also implicated in inflammatory nociception, but mechanisms of their participation have not been elucidated. In the present study, we addressed these mechanisms in the carrageenan-induced mechanical hypernociception, which was determined using a modification of the Randall-Sellito test in rats. Neutrophil accumulation into the plantar tissue was determined by the contents of myeloperoxidase activity, whereas cytokines and PGE2 levels were measured by ELISA and radioimmunoassay, respectively. The pretreatment of rats with fucoidin (a leukocyte adhesion inhibitor) inhibited carrageenan-induced hypernociception in a dose- and time-dependent manner. Inhibition of hypernociception by fucoidin was associated with prevention of neutrophil recruitment, as it did not inhibit the hypernociception induced by the direct-acting hypernociceptive mediators, PGE2 and dopamine, which cause hypernociception, independent of neutrophils. Fucoidin had no effect on carrageenan-induced TNF-
, IL-1β, and cytokine-induced neutrophil chemoattractant 1 (CINC-1)/CXCL1 production, suggesting that neutrophils were not the source of hypernociceptive cytokines. Conversely, hypernociception and neutrophil migration induced by TNF-, IL-1β, and CINC-1/CXCL1 was inhibited by fucoidin, suggesting that neutrophils are involved in the production of direct-acting hypernociceptive mediators. Indeed, neutrophils stimulated in vitro with IL-1β produced PGE2, and IL-1β-induced PGE2 production in the rat paw was inhibited by the pretreatment with fucoidin. In conclusion, during the inflammatory process, the migrating neutrophils participate in the cascade of events leading to mechanical hypernociception, at least by mediating the release of direct-acting hypernociceptive mediators, such as PGE2. Therefore, the blockade of neutrophil migration could be a target to development of new analgesic drugs.
Key Words: hyperalgesia • pain • nociception • cytokines • PGE2
INTRODUCTION
There is consensus in literature that inflammatory hypernociception occurs, at least in part, as a consequence of the sensitization of primary afferent nociceptors. This phenomenon has been attributed to the direct action of hypernociceptive inflammatory mediators (mainly PGs and sympathetic amine) on their receptors present in the nociceptor membrane [1
, 2
]. This lowers nociceptor threshold, with an increase in neuronal membrane excitability [3
]. Nevertheless, it seems that inflammatory stimuli do not directly induce the release of directly acting mediators. Instead, a coordinated cascade of cytokines precedes their release [4
, 5
]. In fact, following the administration of carrageenan in the rat paw, an initial formation of bradykinin occurs, which induces a subsequent release of proinflammatory cytokine cascade [TNF-, cytokine-induced neutrophil chemoattractant (CINC)-1/CXCL1, and IL-1β], triggering the release of PGs and sympathetic amines [6
, 7
].
The mediators involved in the genesis of inflammatory pain also play an essential role in triggering other inflammatory events, including edema and leukocyte migration. For instance, PGs may be crucial for edema formation, and cytokines may be relevant for leukocyte recruitment in several models of inflammation [6 , 8 9 10 11 12 13 ]. However, there are few studies investigating the interdependence of pain with these events, and these few are contradictory. There is evidence that these events may occur independently of each other. For example, nonsteroidal, anti-inflammatory drugs do not interfere with leukocyte migration but inhibit edema formation and pain [8 , 9 , 14 15 16 17 ]. Peripheral analgesics, such as dipyrone, reduce inflammatory pain without affecting edema and leukocyte migration [18 ]. However, it has also been demonstrated that leukocyte migration may be essential for the increase of vessel wall permeability, exudate formation, and pain induction induced by certain inflammatory stimuli [19 20 21 ].
Our group [22
] suggested for the first time the possible nociceptive action of neutrophils. In this study, joint neutrophil accumulation produced intense dog incapacitance when stimulated with a small dose of LPS. Other groups have also investigated neutrophil involvement in the genesis of inflammatory pain. For instance, it was demonstrated that hypernociception induced by leukotriene (LT) the B4; complement 5a factor (C5a) and fMLP depended on neutrophil migration [21
, 23
]. These findings were forgotten in the literature until recently, when it was demonstrated that inhibition of neutrophil migration prevented the hypernociception induced by allergic stimuli in rats [24
]. Considering that activated neutrophils produce and release proinflammatory cytokines, including TNF-, IL-1β, and CINC-1/CXCL1 and mediators such as PGs [25
26
27
28
29
], which are essential to the development of inflammatory hypernociception, it is conceivable to suggest that neutrophils could be a relevant source of hypernociceptive cytokines or alternatively, of the direct-acting hypernociceptive mediators, such as PGs. In the present study, we investigated whether neutrophils played a relevant role in the genesis of inflammatory hypernociception induced by carrageenan and whether neutrophils were a relevant source of hypernociceptive cytokines or direct-acting hypernociceptive mediators.
MATERIALS AND METHODS
Drugs and cytokines
The following materials were obtained from the sources indicated: dopamine, PGE2, bradykinin, and fucoidin (Sigma-Aldrich, St. Louis, MO, USA); indomethacin (Prodome, Campinas, São Paulo, Brazil); rat recombinant (r)IL-1β, rat rTNF-, and rat rCINC-1/CXCL1 (National Institute of Biological Standards and Control, South Mimms, Hertfordshire, UK); and carrageenan (FMC Corp., Philadelphia, PA, USA).
Animals
Male Wistar rats (180–220 g) were housed in temperature-controlled rooms (22–25°C) with access to water and food ad libitum. All experiments were conducted in accordance with National Institutes of Health guidelines for the welfare of experimental animals and with the approval of the Ethics Committee of the Faculty of Medicine of Ribeirão Preto (University of São Paulo, Brazil).
Mechanical hypernociceptive test
In this study, we have used the term hypernociception to describe the decrease in the nociceptive mechanical threshold of an animal [30
]. All experiments were conducted in a double-blind manner, in which the person who injected the solutions was different from the one who made the behavioral assessment. Multiple paw treatments with saline did not alter basal reaction time, which was similar to that observed in noninjected paws. Hypernociception was measured at different times after intraplantar (i.pl.; 100 µl) injection of different stimuli into the hindpaws of rats using the constant pressure rat-paw test, as described previously [31
]. Briefly, a constant pressure of 20 mmHg (measured using a sphygmomanometer) is applied (via a syringe piston moved by compressed air) to a 15-mm2 area on the dorsal surface of the hindpaw and discontinued when the rat presented a typical "freezing reaction." This reaction is comprised of brief apnoea, concomitant with retraction of the head and forepaws, and reduction in the escape movements that animals normally make to free themselves from the position imposed by the experimental situation. Usually, the apnoea is associated with successive waves of muscular tremor. For each animal, the latency of the onset of the freezing reaction is measured before administration (zero time) and at different times after administration of the hypernociceptive agents. The intensity of mechanical hypersensitivity is quantified as the reduction in the reaction time, calculated by subtracting the value of the second measurement from the first [31
]. The intensity of hypernociception was evaluated at indicated times after the administration of carrageenan or inflammatory mediators in the plantar tissue of the rat hindpaw.
Determination of neutrophil accumulation in the hindpaws
Myeloperoxidase (MPO) activity was used as an index of neutrophil accumulation in the rats’ plantar tissues, based on a kinetic-colorimetric assay as described previously [32
]. Approximately 0.5 cm2 plantar tissue was harvested 3 h after the i.pl. injection of inflammatory stimuli. Samples were collected in 50 mM K2HPO4 buffer (pH 6.0) containing 0.5% hexadecyl trimethylammonium bromide and kept at –80°C. Just before the assay, the tissue was homogenized using a Polytron (PT3100) and centrifuged at 13,000 g for 4 min. In our experimental condition (low pH), the MPO assay did not detect the activity of this enzyme in mononuclear cells (data not shown). To prepare the solution for the analysis, 10 µl supernatant was mixed with 200 µl 50 mM phosphate buffer, pH 6.0, containing 0.167 mg/ml O-dianisidine dihydrochloride and 0.0005% hydrogen peroxide. The solution was analyzed by spectrophotometry for MPO activity determination at 450 nm (Spectra max) with three readings in 1 min. The MPO activity was compared with a standard curve of neutrophils obtained from rats’ blood. The results were presented as number of neutrophils x106/mg tissue.
Cytokine measurements
At 3 h after carrageenan i.pl. injection, animals were terminally anesthetized, and the plantar tissues (
0.5 cm2) were removed from the injected and control paws (saline and naïve). The time-point for cytokine measurement was defined based on the time-course measured of cytokines induced by carrageenin [33
]. It was demonstrated that 3 h after carrageenin is the peak of cytokine production [33
]. The samples were triturated and homogenized in 500 µl of the appropriate buffer (PBS containing 0.05% Tween 20, 0.1 mM PMSF, 0.1 mM benzethonium chloride, 10 mM EDTA, and 20 kallikrein IU Aprotinin A), followed by a centrifugation of 10 min/2000 g. The supernatants were stored at –70°C until further analysis. The levels of TNF-, IL-1β, and CINC-1/CXCL1 were evaluated using sandwich ELISA. ELISA kits for TNF-, IL-1β, and CINC-1/CXCL1 were from the National Institute for Biological Standards and Control (Potters Bar, UK). Briefly, sheep polyclonal anti-rat TNF-, IgG-raised TNF- (2.0 µg/ml); sheep polyclonal anti-rat IL-1β, IgG-raised IL-1β (2.0 µg/ml); or sheep polyclonal anti-rat CINC-1, IgG-raised CINC-1/CXCL1 (3.0 µg/ml), diluted in 50 µl PBS buffer, was used to coat microtiter plates (Nunc Maxisorb, Roskilde, Denmark). Blocking of nonspecific binding sites was accomplished by incubating plates with PBS containing 2% BSA for 90 min at 37°C. After incubation (4°C, overnight) and washing the plates in assay buffer (0.01 M phosphate, 0.05 M NaCl, 0.1% Tween 20, pH 7.2), 50 µl standard (CINC-1, TNF-, IL-1β, or sample) was added to each well and incubated overnight at 4°C. After washing the plates, 50 µl biotinylated rabbit polyclonal anti-rat TNF- (1:2000 with assay buffer plus 1% normal sheep serum) and rabbit anti-rat IL-1β (1:2000) or sheep polyclonal anti-rat CINC-1 IgG (diluted 1:500) antibody for 30 min at 37°C were added to the plates and incubated for 1 h at room temperature. After incubation, the plates were washed again, and 50 µl avidin–peroxidase conjugate (1:5000 dilution, Dako, Carpinteria, CA, USA) was added to all the wells, and the wells were incubated for 30 min with 50 µl substrate (40 µg well–1, orthophenylenediamine dihydrocloride, Sigma-Aldrich). After color development, the reaction was stopped with the addition of sulfuric acid (1 M). Absorbance was measured at 490 nm. These ELISA methods consistently detected TNF-, IL-1β, and CINC-1 levels over 20 pg/ml and did not cross-react with other cytokines. The results are expressed as picograms (pg) of each cytokine per paw. As a control, the concentrations of these cytokines were determined in naïve mice and in animals injected with saline [33
34
35
].
Measurement of PGE2 in paw skin
The plantar tissues were collected 2 h after i.pl. injection of IL-1β (0.5 pg/paw) or saline as described above. This time-point is the peak of IL-1 β-induced PGE2 production in the rat paw (data not shown). The paws were injected with indomethacin (100 µg/paw) 10 min before tissue retrieval to block PGE2 production during tissue processing. The PGE2 was extracted from plantar tissue and determined by radioimmunoassay (RIA) [36
]. Briefly, the plantar tissue samples were homogenized in a mixture of 3.0 ml extraction solvent (isopropanol/ethyl acetate/0.1 N HCl, 3:3:1) and 3.0 ml distilled water. Also, the solution contained 20 µg/ml indomethacin. Homogenates were centrifuged at 1500 g for 10 min at 4°C. The organic phase was aspirated and evaporated to dryness in a centrifugal evaporator. The pellet was reconstituted in 500 µl 0.1 M phosphate buffer (pH 7.4) containing 0.8% sodium azide and 0.1% gelatin. Concentration of PGE2 in these samples was then measured by RIA by using a commercially available kit. The results are expressed as pg PGE2 per paw.
Neutrophil isolation and culturing
Rats’ blood (10–12 ml) was collected through cardiac puncture into heparinized syringes. Neutrophils were isolated from heparinized blood via Ficoll-Paque (Pharmacia, Peapack, NJ, USA) gradient centrifugation. The erythrocyte/granulocyte pellet was diluted 1:1 with normal saline. Erythrocytes were sedimented by 3% dextran (Sigma-Aldrich) in 0.9% saline and incubated for 1 h. Supernatants were collected and centrifuged twice 2x 1000 g for 10 min at 4°C. Remaining RBCs were lysed in distilled water. The freshly isolated neutrophils were resuspended in modified RPMI 1640 (Sigma-Aldrich) containing 10% heat-inactivated FBS (Sigma-Aldrich), 100 U/ml penicillin, 100 µg/ml streptomycin, and 300 µg/ml glutamine (Sigma-Aldrich). The neutrophil preparation routinely contained 95% neutrophils, as identified by the Giemsa stain, and were found to be 98% viable by the Trypan blue exclusion technique [37
]. These cultured neutrophils were incubated with vehicle (Tris/HCl) or indomethacin (10 µg/mL) for 20 min and subsequently stimulated with IL-1β (100 ng/ml) for 4 h. PGE2 concentration in the supernatant was determined by RIA [36
].
Statistical analyses
The results are representative of two independent experiments as the mean ± SEM (n=5 per group in each individual experiment). One-way ANOVA followed by Bonferroni’s t-test was performed to evaluate the differences between responses. Statistical differences were considered to be significant at P < 0.05.
RESULTS
Neutrophils play a critical role in carrageenan-induced mechanical hypernociception
It is well established in literature that fucoidin inhibits leukocyte migration [38
39
40
41
]. As the leukocyte influx induced by carrageenan in the time-frame of our experiments is composed almost exclusively by neutrophils (>95%) [42
43
44
], fucoidin was used to evaluate the role of neutrophil migration in the induction of inflammatory hypernociception. The doses of fucoidin were based in previous studies [37
, 39
]. As shown in Figure 1
, fucoidin given 5 min before i.pl. injection of carrageenan (100 µg/paw) inhibited mechanical hypernociception in a dose (6.6, 20, and 60 mg/Kg/i.v., diluted in saline)- and time (1–5 h)-dependent manner (Fig. 1A
and 1C)
. This effect on inflammatory hypernociception seems to be closely associated with the capacity of fucoidin to inhibit carrageenan-induced neutrophil migration to the plantar tissue, as evaluated by MPO activity assay (Fig. 1B
and 1D)
. It is noteworthy that fucoidin treatment did not affect the basal nociceptive threshold of the animals (data not shown). Reaction time baseline was 31.5 ± 0.1 s (means±SEM; n=36) before injection of the hypernociceptive agents.
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Figure 1. Fucoidin dose-dependently inhibits mechanical hypernociception and neutrophil migration to the plantar tissue. Rats were treated with fucoidin (6.6, 20, and 60 mg/Kg, i.v.) or vehicle [saline (Sal)] 10 min before the i.pl. injection of carrageenin (Cg; 100 µg/paw) or saline. The hypernociceptive responses were evaluated 3 h after stimuli, followed by the collection of the plantar hind-paw tissue for MPO analysis (A and B, respectively). The effect of fucoidin (Fuc; 20 mg/Kg i.v.) on hypernociception and neutrophil migration was also evaluated 1, 3, and 5 h after carrageenan administration (C and D, respectively). *, P < 0.05, compared with the control saline paw-injected group; #, compared with the vehicle-treated group; ANOVA One-way followed by Bonferroni’s t-test (n=5).
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Figure 2. Effect of fucoidin upon hypernociception and neutrophil migration induced by PGE2 or dopamine. Rats were treated with fucoidin (20 mg/Kg, i.v.) or vehicle (saline) 10 min before the i.pl. injection of PGE2 (100 ng), dopamine (Dopa; 10 µg), carrageenin (100 µg/paw, positive control), or saline (negative control). The hypernociceptive responses were evaluated 3 h after stimuli, followed by the collection of the plantar hind-paw tissue for MPO analysis (A and B, respectively). *, P < 0.05, compared with the control saline paw-injected group; #, compared with the vehicle-treated group; ANOVA One-way followed by Bonferroni’s t-test (n=5).
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, IL-1β, and CINC-1/CXCL1, in the site of inflammation is essential for the development of inflammatory hypernociception [4
]. Treatment with fucoidin (20 mgKg, 5 min, i.v.) did not affect the local production of TNF-, IL-1β, and CINC-1/CXCL1 induced by i.pl. injection of carrageenan (Fig. 3A
3B
3C
). Thus, although neutrophils play a role in mediating carrageenan-induced inflammatory hypernociception, these cells are not relevant for carrageenan-induced cytokine release.
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Figure 3. Fucoidin does not affect carrageenan-induced cytokine production in the plantar tissue. Rats were treated with fucoidin (20 mg/Kg, i.v.) or vehicle (saline) 10 min before the i.pl. injection of carrageenin (100 µg/paw) or saline. At 3 h after the injection of stimuli or naïve animals (noninjected animals), the hind-paw plantar tissue was collected in the appropriate buffer containing protease inhibitors, and the samples were processed for cytokine level analysis. *, P < 0.05, compared with the saline paw-injected group; ANOVA One-way followed by Bonferroni’s t-test (n=5).
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(1 pg/paw), IL-1β (0.5 pg/paw), and CINC-1/CXCL1 (100 pg/paw; Fig. 4A
). In addition, bradykinin, TNF-, IL-1β, and CINC-1/CXCL1 induced neutrophil migration to the plantar tissue, which was inhibited by the pretreatment with fucoidin (Fig. 4B)
. Thus, the local cytokine cascade triggered by carrageenan or bradykinin is an event that precedes neutrophil influx, and cytokines act in a neutrophil-dependent manner to induce hypernociception.
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Figure 4. Effect of fucoidin on hypernociception and neutrophil migration induced by bradykinin, TNF-, IL-1β, and CINC-1. Rats were treated with fucoidin (20 mg/Kg, i.v.) or vehicle (saline) 10 min before the i.pl. injection of saline, bradykinin (BK; 500 ng/paw), TNF- (1 pg/paw), IL-1β (0.5 pg/paw), and CINC-1/CXCL1 (100 pg/paw). The hypernociceptive responses were evaluated 3 h after stimuli, followed by the collection of the plantar hind-paw tissue for MPO analysis (A and B, respectively). *, P < 0.05, compared with the control saline paw-injected group; #, compared with the vehicle-treated group; ANOVA One-way followed by Bonferroni’s t-test (n=5).
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Figure 5. IL-1β induces PGE2 production depending on neutrophils. Rats were treated with fucoidin (20 mg/Kg, i.v.) or vehicle (saline) 10 min before the i.pl. injection of saline or IL-1β (0.5 pg/paw). At 2 h after stimuli injection, the plantar hind-paw tissue was collected for PGE2 level measurements (A). Neutrophils were isolated and stimulated in vitro with IL-1β (100 ng/mL) or RPMI and treated with indomethacin (Indo; cyclooxygenase inhibitor, 10 µg/mL) or Tris/HCl (B). After 4 h, the supernatant was collected for PGE2 measurement by RIA. *, P < 0.05, compared with the saline paw-injected group and medium-treated group; #, compared with the vehicle-treated group; ANOVA One-way followed by Bonferroni’s t-test (n=5).
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Generally, neutrophils are the first line of defense of the immunological system against pathogens. However, in several inflammatory diseases, such as rheumatoid arthritis, they represent a potential cause of tissue damage. Indeed, the interaction of recruited neutrophils in the site of inflammation with resident cells, local inflammatory mediators, and/or extracellular matrix may lead to the production of several other mediators, including cytokines/chemokines, degrading enzymes, oxygen and nitrogen species, and metalloproteases that may further amplify the inflammatory response and injure surrounding tissue [28 , 45 46 47 48 49 ]. Neutrophil-derived mediators may also contribute to edema formation, fever, and production of acute-phase proteins [19 , 50 ]. In the present study, we have demonstrated that neutrophils are involved in the genesis of inflammatory hypernociception. Moreover, we demonstrated for the first time that the hypernociceptive actions of neutrophils were not associated with the release of hypernociceptive cytokine by migrating neutrophils. On the contrary, effects of hypernociceptive cytokines depend on neutrophil migration and the ability of these cells to release direct-acting mediators such as PGE2.
The pronociceptive action of neutrophils was first suggested almost 30 years ago [22 ]. It was observed that neutrophils present in the joints of dogs enhanced the articular nociception promoted by LPS administration. In subsequent studies, Levine et al. [21 , 23 ] showed that i.pl. administration of LTB4, C5a, or fMLP in rat paw promoted mechanical hypernociception, which depended on polymorphonuclear cell migration. Additionally, the thermal hypernociception induced by nerve growth factor, a well-recognized pronociceptive mediator, was inhibited in neutrophil-depleted rats [51 , 52 ]. Indirect evidence for the involvement of neutrophils in inflammatory hypernociception was demonstrated in zymosan-induced joint inflammation. In this model, the articular movement incapacitation was inhibited by the LT synthesis inhibitor, which also decreased the neutrophil accumulation into the joints [53 ]. Furthermore, Lavich et al. [24 ] recently showed that neutrophils played a crucial role in thermal hypernociception induced by an allergic stimulus. The inhibition of neutrophil migration with fucoidin or antineutrophil antiserum reduced hypernociception in OVA-induced allergic response [24 ]. The neutrophils also present an important role in the establishment of hyperalgesia as a result of partial nerve injury [54 ]. In humans, there is also an association between the hyperalgesic effects of LTB4 and neutrophil migration kinetics [55 , 56 ]. In agreement, neutrophil infiltration into the joint of arthritis patients precedes clinical signs of inflammation and is predictive of pain [56 ]. Although these studies proposed the involvement of neutrophils on the nociceptive process, they did not address the mechanism by which these cells mediate inflammatory hypernociception.
In the present study, fucoidin was used as a pharmacological tool to evaluate the role of neutrophils in the genesis of inflammatory hypernociception. This drug inhibits leukocyte migration, as it binds to L- and P-selectins and consequently, inhibits the leukocyte rolling [38 , 41 ]. The treatment of animals with fucoidin inhibited in a dose-dependent manner the mechanical hypernociception induced by carrageenan. This observation was closely associated with the inhibition of neutrophil migration to s.c. plantar tissue of rats treated with fucoidin. Besides the direct inhibition of neutrophil migration by binding to L- and P-selectins, it has been suggested that fucoidin presents other pharmacological actions, including the stimulation of NO production [57 , 58 ]. Considering that NO has a peripheral antinociceptive action on the hypernociception induced by direct-acting mediators [59 , 60 ], we tested the effect of fucoidin on hypernociception induced by PGE2 and dopamine. Supporting our hypothesis that the inhibition of neutrophil migration is responsible for the antihyperalgesic effect of fucoidin, the drug did not inhibit the mechanical hypernociception induced by these direct-acting hypernociceptive mediators. Moreover, PG and dopamine in doses that cause mechanical hypernociception did not produce significant neutrophil migration to the rat paw.
We have demonstrated that mechanical hypernociception induced by carrageenan in rats is mediated by the release of a cascade of cytokines initiated by bradykinin, which is rapidly generated when plasma components reach extravascular tissue [7
]. Although neutrophils are able to produce and release cytokines, including TNF-, IL-1β, and chemokines, in vitro and in vivo [26
, 28
, 61
62
63
64
], in the present study, we demonstrated that the carrageenan-induced increase of cytokines production in the rat plantar tissue was independent of neutrophil migration. It is likely that the cytokines (TNF-, IL-1β, CINC-1/CXCL1) involved in the mediation of hypernociception might be released by resident cells such as macrophages, mast cells, and fibroblasts. Indeed, these cells are able to release cytokines when activated by inflammatory stimuli [65
66
67
68
69
]. It is important to mention that neutrophils could be responsible for the release of pronociceptive cytokines in other models of inflammation. In this regarding, the depletion of rat neutrophil population with antineutrophil antibody prevented the increase of IL-1β levels in the rat plantar tissue stimulated with CFA [64
]. Moreover, recently, we demonstrated that inhibition of neutrophil migration by a new CXCR2 antagonist reduced cytokine production during adjuvant-induced arthritis in rats [70
]. The difference in experimental models may explain the apparent contradiction.
Although the recruited neutrophils are not the source of hypernociceptive cytokines, at least in our inflammatory model, it was observed that neutrophil migration was important for the hypernociceptive effect of bradykinin and cytokines. These results led us to suggest that neutrophils were mediating the release of direct-acting hypernociceptive mediators (e.g., PGs and sympathetic amines) triggered by the cytokine cascade. In fact, we detected that inhibition of neutrophil migration by fucoidin inhibited the production of PGE2 in the rat paws stimulated by IL-1β. In addition, incubation of cultured neutrophils with IL-1β increased the production of PGE2. This is consistent with other studies that show neutrophils produce PGs when stimulated with cytokines [25 , 71 ]. Therefore, it seems that during the inflammatory process, cytokines mediate the recruitment of the neutrophils and activate them to produce final hypernociceptive mediators such as PGE2. These results do not discard the possibility that neutrophils contribute to inflammatory hypernociception by another mechanism. In support of the latter possibility, fucoidin inhibited neutrophil migration and hypernociception induced by CINC-1/CXCL1, whose effect does not depend on prostanoid production but instead, depends on the release of sympathetic amines [72 ]. Neutrophils could produce or facilitate the production of inflammatory mediators, including degrading enzymes, free radicals, metalloproteases, and hydrogen protons, which may then contribute to the release of direct-acting hypernociceptive mediators [73 74 75 76 77 ]. There is also evidence that human fMLP-stimulated neutrophils produce 15-hydroxyeicosatetraenoic acid, a lipid metabolite produced by the 15-lipoxygenase enzyme, which has a strong hypernociceptive action [23 ]. However, in the inflammatory model used in the present study (carrageenan-induced inflammation), inhibitors of 15-lipoxygenase did not alter carrageenan-induced hypernociception (data not shown). Furthermore, the neutrophil-derived proteases can also be involved in the generation of kinin (e.g., bradykinin) from plasma kininogens, which could synergize with other hypernociceptive mediators, contributing to the nociceptor sensitization and consequently, to establishment of hypernociception [78 79 80 ]. Although these results clearly suggest the importance of neutrophil in the genesis of inflammatory hypernociception, having only the presence of neutrophil in the inflammatory focus is not sufficient for the onset of the hypernociception. Neutrophils need to be activated [22 , 23 ]. For example, recruitment of neutrophils using glycogen did not cause hypernociception, but when neutrophils were activated with LPS or C5a, the hypernociceptive state was established [22 , 23 ].
Although the present data suggest that neutrophils are essential for the genesis of inflammatory pain, there is also evidence that nociception can counteract the neutrophil migration to the inflammatory site and consequently, reduce the extension of the inflammatory process. For instance, it was demonstrated that activation of nociceptive neurons induces shedding of L-selectin from circulating neutrophils in vivo and that this shedding prevents neutrophil accumulation into joints induced by bradykinin [81 ]. These findings indicate other interdependence between the inflammatory events.
In conclusion, the present study demonstrated the importance of neutrophils for the genesis of inflammatory hypernociception. The pronociceptive action of neutrophils did not depend on their ability to produce cytokines, but instead, the pronociceptive action of cytokines depended on the production of the final hypernociceptive mediators by neutrophils, such as PGE2 (Fig. 6 ). These results reinforce the role of neutrophils in hypernociceptive states and suggest that targeting of neutrophils may be useful therapeutically in the control of inflammatory pain.
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Figure 6. Schematic representation of the role of neutrophils in the rat-paw inflammatory hypernociception genesis. Neutrophils migrate in response to cytokines, and this effect is inhibited by the treatment with fucoidin. In the site of inflammation, neutrophils produce PGE2 that sensitizes the nociceptor (primary afferent neuron). Furthermore, sympathetic neurons release sympathetic amines (SA) that also sensitize the nociceptor fibers during inflammation. Neutrophils also contribute to this sympathetic component of inflammatory hypernociception, but the precise mechanisms remain to be determined.
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ACKNOWLEDGEMENTS
This work was supported by grants from Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP; Brazil) and Conselho Nacional de Pesquisa (CNPq; Brazil). T. M. C. (Ph.D. student) is a recipient of fellowships from FAPESP, and W. A. V. is a recipient of a postdoctoral fellowship from FAPESP. The authors gratefully acknowledge the technical assistance of Sérgio R. Rosa and Giuliana B. Francisco.
Received September 24, 2007; revised November 30, 2007; accepted December 5, 2007.
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